ADP-ribosylation factors (ARFs) are multi-functional, multi-domain, proteins that regulate vesicular trafficking in the ER and Golgi (and elsewhere). ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs). In addition to a family of ~200-kDa GEPs that are inhibited by BFA (a drug that inhibits protein secretion and causes reversible disintegration of Golgi cisternae) is a family of ~55-kDa GEPs that are BFA-resistant. All have so-called Sec7 domains of ~200 amino acids that are responsible for GEP activity. Cytohesin-1, cloned by its interaction with integrin, had been characterized by the group as a BFA-insensitive ARF GEP. Predicted amino acid sequences for cytohesin-2 (83% identical) differed only in the presence or absence of a single glycine. The group demonstrated the existence of two mRNA isoforms that differ in the same way for each of three know cytohesins and identified a new cytohesin-4 with only one mRNA. To elucidate mechanism(s) that control the single glycine insertion, genes for cytohesin-1 and -4 were compared, revealing remarkable similarity of structure except for an extra 3-bp exon in cytohesin-1. The extra glycine is expected to have major effects on function, but probably not on GEP activity, since removal of the PH domain abolishes cytohesin activation by PIP2 but not GEP activity. Although, cytohesin-4 resembles the others in its GEP activity and gene structure, it differed in distribution in specific lymphocyte subpopulations, suggestive of different functions.